Asymmetric divisions contribute to cell fate specification during embryonic development and stem cell maintenance. Spindle alignment with a polarized axis is essential for this process, and spindle position is also crucial in symmetrically dividing cells to maintain proper cellular arrangements. Disregulation of spindle positioning and polarity has been implicated in cancer. This proposal addresses how multiple polarity cues coordinate to produce proper spindle alignment, using the early Caenorhabditis elegans embryo. Asymmetric division of the one-cell embryo relies on a conserved pathway in which the PAR polarity proteins regulate cortical asymmetry of the G?/GPR/LIN-5 complex; this complex recruits the microtubule motor dynein to generate pulling forces that orient spindles. LET-99, a DEPDC1 family protein, is localized in an asymmetric cortical band pattern on the anterior/posterior (AP) axis by the PAR proteins, and LET-99 in turn restricts the localization of GPR/LIN-5 to generate asymmetric pulling forces. Similar PAR and LET-99 domains are reestablished in the P1 daughter of the first division. P1 divides into P2 and EMS, which both divide asymmetrically, but only P2 exhibits AP PAR domains. The EMS division is regulated by Wnt and Mes/Src signaling pathways, which polarize the cell and align the spindle on the AP axis. LET-99, LIN-5 and the PAR proteins are present in EMS, but here the PARs are polarized along the inner/outer instead of the AP axis. Our recent studies using fast-inactivating temperature sensitive alleles reveal that LET-99 has a primary role in spindle positioning in many cells of the early embryos, and acts in the Mes/Src pathway in EMS. We also identified roles for LIN-5 and the PAR-1 related kinase PIG-1 in EMS. Our observations support our central hypothesis that LET-99 acts downstream of multiple polarity cues to inhibit the localization or activity of LIN- 5 and thus regulate spindle position in different cell types. In Aim 1, we will elucidate mechanisms within the Mes/Src pathway using genetic analysis to test the hypothesis that PIG-1 acts upstream or parallel to LET-99. We will also determine if LET-99, LIN-5, PIG-1 and/or DNC (dynactin, a dynein regulator) are asymmetrically localized in EMS, and test the hypothesis that LET-99 prevents recruitment of cortical LIN-5. This aim will use photo-activated fluorescent proteins to resolve cortical asymmetry in a multicellular context. In Aim 2, we will use a combination of genetics and biochemistry to define the role of CED-10/RAC in the Mes/Src pathway and distinguish between several hypothesis for how LET-99 and CED-10 interact. In Aim 3, we will use live imaging studies and temperature sensitive mutants to test several models for P1 polarity reestablishment. The regulation of the second division is little studied, and LET-99, LIN-5 and PIG-1 are the first intermediates for spindle positioning in the Mes/Src pathway to be analyzed. This research will significantly advance our knowledge of these pathways. Because the pathway components are conserved, the results will be relevant to asymmetric division and spindle positioning in many systems.